Ribosome binding to endoplasmic reticulum

Christopher M. Sanderson, Adam J. Savitz, David I. Meyer
1991 Cell Biophysics  
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER) and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1 u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here we examined the role of ribosome binding and
more » ... inding and depurination activity on inhibition of the UPR using mRTA mutants. An active site mutant with very low depurination activity, which bound ribosomes as wild type RTA, did not inhibit HAC1 u mRNA splicing. A ribosome binding mutant, which showed reduced binding to ribosomes, but retained depurination activity inhibited HAC1 u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. Ribosome binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1 u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of HAC1 u mRNA. We show that HAC1 u mRNA is associated with ribosomes and does not get processed on depurinated ribosomes thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1 u mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1 u mRNA that is associated with ribosomes. Ricin is a type II ribosome inactivating protein (RIP) produced by the castor bean plant Ricinus communis. Due to its toxicity and ease of isolation, ricin has been classified as a category B select agent [1]. Ricin is more toxic to cancer cells than healthy cells and has been used as the toxic component of immunotoxins [2] [3] [4] . The holotoxin is comprised of an enzymatically active ricin toxin A chain (RTA) linked to ricin toxin B chain (RTB) by a disulfide bond. RTB facilitates entry of the holotoxin into the cell by binding to glycoproteins or glycolipids on the cell surface. After endocytosis, a small fraction of ricin is transported to the endoplasmic reticulum (ER) via retrograde transport. Upon entry into the ER, the disulfide bond of the holotoxin is reduced, allowing RTA to separate from RTB. Reduction of the disulfide bond partially unfolds RTA and allows it to cross the ER membrane. RTA is thought to exploit the ERassociated degradation (ERAD) pathway to enter the cytosol [5, 6] . A fraction of RTA escapes the ubiquitin mediated-degradation in the cytosol ultimately reaching ribosomes [7] . RTA inhibits protein synthesis by removing an adenine from the sarcin/ricin loop (SRL) of the 28S rRNA. This depurination event halts translation at the elongation step leading to cell death. http://www.jbc.org/cgi/
doi:10.1007/bf02989875 fatcat:jjl247jcm5c3vbmob442ei5uce